Pathogen surrogates based on encapsulated tagged DNA for verification of sanitation and wash water systems for fresh produce

ABSTRACT

A pathogen surrogate, formed by a DNA tag or bar code and a carrier, is described for use in the validation and verification of sanitation, such as in food processing operations and for wash water systems for fresh produce. The carrier material is selected so that the pathogen surrogate mimics the behavior of a pathogen when subjected to a sanitation operation. One or more surrogates can be introduced in to an environment, which is then subjected to sanitation process, followed by a detection process using the DNA tag of the surrogate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.62/200,456 filed on Aug. 3, 2015, which is hereby incorporated in itsentirety by this reference.

BACKGROUND OF THE INVENTION

The following disclosure relates generally to the validation andverification of sanitation, disinfection and wash processes and, morespecifically, to the use of tagged DNA for such verification.

Microorganisms are biological entities that can be beneficial orhazardous to humans and to the food manufacturing industry. Bacteriarepresent the largest group of microorganisms. Most bacteria areharmless; in fact, some provide benefits to humans by protecting theskin and nasal passages and also aid in the digestion process. Some alsobenefit the food industry, when they are used for production of cultureditems, such cheese, yogurt, and fermented sausage. However, they canpose a threat to humans and to the industry when they result infoodborne illness and food spoilage. Microbiological contamination offood may result in product spoilage, reduction in shelf life, orfoodborne illness.

The time used in cleaning a food plant is time away from production.Yet, it is production that is the purpose of a food plant, providedproduction is wholesome, unadulterated, and of a quality level thatpeople will continue to buy the finished product. Without a sanitationprocess in a food plant, it is likely that none of these expectationswill be met. Sanitation is basic to food safety and quality. It is avital segment of an integrated food safety system with strong links toregulatory compliance, quality, Hazard analysis and critical controlpoints (HACCP), (Good manufacturing practices) GMPs, and pest control.The process of sanitation has many facets that make it vital in a foodplant, not the least of which is that it allows food companies to meetregulatory standards. Of course, the primary function is to removecontaminating soils, prevent film buildup, and prepare food contactsurfaces for production. It is also necessary to prevent insect androdent infestation and harborage by removing sources of attraction andnutrition. Effective sanitation also plays an important role inpreventing allergen cross-contamination and foreign material inclusions.The benefits of effective sanitation are production of safe product,improved product shelf life, and reduction of off-flavor, odor, andcolor. To an extent, it will also prevent equipment deterioration andincrease production efficiency. Finally, it can be a source of pride andmorale to employees who prefer to work in a location that is clean.

Contamination can also occur in wash water systems used in fresh produceprocessing. Food agriculture occurs mostly outdoors, so the foodproducts are susceptible to contamination from the soil in which it isgrown, various arthropods that co-inhabit the environment, and excretafrom birds and other animals. Thus, most fruits, vegetables, and nutssold as fresh produce in the U.S. are washed by the packer/shipper priorto distribution. This washing is effective at removal of grosscontamination, which may be associated with fungi and bacteria that cancause disease or accelerate spoilage. However, microbiologicalcontamination is comparatively tenacious and may not be adequatelyreduced by washing under conditions that preserve the product. In termsof HACCP, washing is not considered a kill step. In fact, washingproduce creates a risk of inadvertent cross-contamination, as thecontaminant breaks down and spreads microorganisms throughout the wash.

In order to reduce the likelihood of microbial contamination of produce,wash water (and wash water additives) should not only remove attachedmicrobes from the surface of the produce, but also prevent microbialdeposition from the wash water to the produce. Validation of thesemethods requires experiments using target organisms. However,intentional contamination of industrial food handling equipment withpathogens and spoilage organisms is counterproductive. Non-pathogenicbacteria, which have similar responses to specific food processes as thepathogenic bacteria, have been developed for food processors to validatea process in-plant, without the use of actual pathogens. However, theindustry is resisting the use of live bacteria in food processingplants, whether they are considered pathogenic or not. This is partlybecause of experiences with organisms which were considerednon-pathogenic but caused reported illnesses, and partly because DNAfragments from these organisms may be detected during routine microbialtesting and result in false positives with highly detrimental economiceffects.

SUMMARY

In a first set of aspects, a product includes a DNA bar code and anon-toxic pathogen surrogate carrier. The DNA bar code is combined withthe surrogate carrier to form a pathogen surrogate, where the pathogensurrogate is degradable under a sanitation process.

In another set of aspects, a method of providing a pathogen surrogateincludes receiving a carrier formed of a non-toxic substance andcombining a DNA bar code with the carrier to form a pathogen surrogate.A benchmarking operation is performed to determine whether pathogensurrogate behaves similarly to a first pathogen when subjected to asanitation operation.

In a further set of aspects, a method of testing the efficacy of asanitation process includes introducing a first pathogen surrogateformed of a DNA bar code encapsulated in a non-toxic pathogen surrogatecarrier. A sanitation operation is subsequently performed. A detectionoperation determines the amount of the first pathogen surrogate presentafter performing the sanitation operation.

All patents, patent applications, articles, books, specifications, otherpublications, documents and things referenced herein are herebyincorporated herein by this reference in their entirety for allpurposes. To the extent of any inconsistency or conflict in thedefinition or use of a term between any of the incorporatedpublications, documents or things and the text of the present document,the definition or use of the term in the present document shall prevail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a pathogen surrogate formed of DNA bar code and acarrier material.

FIG. 2 presents a method of providing a pathogen surrogate.

FIG. 3 illustrates the use of pathogen surrogates in a sanitationprocess.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The DNATrax technology described herein is already used in aerosolformulations for simulation of bio-threat microparticles in modelingterrorist attacks, and for tracking and quantifying particulatemigration. See for example, U.S. Pat. No. 8,293,535 and U.S. Application2014/0057276 on the formation of DNA tagged microparticles in thiscontext. Related U.S. Application 2014/0272097 discloses DNA marking ofitems for authenticating the items, while U.S. patent application Ser.No. 14/599,315 extends the use of such materials to the problem oftracing the origin of food products. However, none of these functionsrelate to the use of such DNA bar code or tag (“bar code” and “tag” areused interchangeably herein) to the verification of sanitation and washwater systems for fresh produce. Although the following discussion willbe mainly in the context of food processing, it is also relevant inother application such as disinfection and sanitation of healthcarefacilities, medical devices, aerospace missions, and other applications.

In the sanitation context, the particle properties can be adjusted tosimulate the behavior of living organisms (often, although notnecessarily, pathogenic) under specific conditions, such as pH,temperature, etc. Chemical reactions between the carrier materials andthe sanitizers promote the destruction of the particles at rates thatare either similar or correlate to those of the target pathogens.Further, the surface properties of the particles can be adjusted toadhere to processing or product surfaces in a way that is similar tothat of the target organisms. Some examples of pathogens where thesetechniques could be applied in the food processing context and moregenerally, such as in a healthcare settings, include salmonella,methicillin-resistant Staphylococcus aureus (MRSA), and Clostridiumdifficile, tuberculosis (MTB, TB), hepatitis B, and humanimmunodeficiency virus (HIV), among others.

Sanitation Validation and Verification

No sanitation system is complete without proof of effectiveness. Forindustries concerned with safe and reproducible production, the initialrigorous proof of effectiveness for a new process is called validation.A proof for effectiveness of the same process with slightly differentequipment (e.g., after maintenance) or a slightly different product(e.g., a new lot of raw material) is called verification. Verificationis typically performed more frequently than validation and is typicallysomewhat less rigorous.

Validation and verification may be done in several ways, from simple andrelatively inexpensive to slightly more expensive and complex. The leastexpensive and easiest to implement is visual or organoleptic examinationof the post-sanitation and pre-operation environment. Organolepticpre-op inspection is normally required as part of the plant's SOP, andthere is no regulatory requirement to incorporate other investigativetools. However, added investigative tools and documentation provideextra insight into the thoroughness of the sanitation process. Adenosinetriphosphate (ATP) or bioluminescence measurement is an extremelyeffective, relatively inexpensive tool that many food manufacturersemploy for rapid verification feedback about sanitation. Microbiologicaltesting is a tool used by many companies as a means of both verificationand validation of sanitation.

Organoleptic testing involves direct sight, smell, and touch. This istypically performed as verification during pre-op inspection, followingthe sanitation process. It involves inspection of the equipment,obviously using the sense of sight to look for any indications of foodmaterial left on equipment, such as grease, dough, or produce, dependingon the products being handled in the plant. The sense of smell mayidentify hidden material and is especially sensitive for detection ofputrescence. Touch can detect thin films that are slippery, sticky, orrough in texture.

Rapid microbial detection is becoming increasingly essential to manycompanies in the pharmaceutical, clinical, and in the food and beverageareas. Faster microbiological methods are required to contribute tobetter control of raw materials as well as finished products. Rapidmethods can also provide better reaction time throughout themanufacturing process. Rapid technologies provide companies with costsavings and speed up product releases.

Despite clear disadvantages, traditional microbial detection methods arestill widely used. Current methods require incubation of products inliquid or solid culture media for routinely 2 to 7 days before gettingthe contamination result. This necessary, long incubation time is mainlydue to the fact that stressed microorganisms found in complex matricesrequire several days to grow to visible colonies to be detected.Moreover, this incubation period can be increased up to 14 days inspecific applications like sterility testing for the release ofpharmaceutical compounds. This delay can cause two significant problems.One is that fresh foods have a limited shelf life, not much longer thanthe 7 to 14 days required for culture testing. So these products must beshipped prior to test results. The other problem is that, in the case ofan outbreak of foodborne disease, victims get sick and die during those7 to 14 days before the product is confirmed to be contaminated and arecall is accomplished.

Although these techniques have advantages including technicalrobustness, low skill and training requirements, the use of inexpensivematerials and their acceptability to the regulatory authorities, themajor drawback is the length of time required to get microbiologicalresults. Thus, in light of the growing demand for rapid detectionmethods, various alternative technologies have been developed. In thefield of rapid micro-organisms detection, ATP bioluminescence based onluciferin/luciferase reaction has garnered great interest.

Adenosine triphosphate (ATP) is found in all living organisms and is anexcellent marker for viability and cellular contamination. Rapiddetection of ATP through ATP-luminescence technology is therefore amethod of choice to replace traditional methods and significantlyshortens the time to detection. However, ATP has a number ofdisadvantages, including: It does not easily distinguish between ATPfrom microorganisms, animals, and plants; luminescence from product cangenerate spurious ATP bioluminescence readings; the presence ofdetergents, sanitizers, or other chemicals also can affect the readings;and it is not sensitive for spore detection, where ATP is very low.

Use of Surrogates in Sanitation

One of the practical limitations of process validation is that theactual pathogens cannot be taken in to a food-processing establishmentto verify a specific process. While laboratory research can be used as areference point, it is not a true substitute for actual, in-plantprocess validation. The availability of non-pathogenic bacteria, whichhave similar responses to specific food processes as the pathogenicbacteria, offers the food processor the ability to validate a processin-plant, without the use of the actual pathogens. For example,coliforms have been used as process indicators for pasteurization in thedairy industry.

The term surrogate is used to indicate a substitute for an item ofinterest. In the context of environmental microbiology and health riskassessment, a surrogate is defined as an organism, particle, orsubstance to study the fate of a pathogen in a specific environment.Surrogates may be released into the environment to evaluatedecontamination or disinfection achieved within a food processing plant.In this case, the surrogate should not damage or contaminate theequipment or processes, or at least the equipment can be easilydecontaminated. Surrogates should be avoided if they could causeundesirable biofilm formation, taste, odor, physicochemical changes, oran opportunistic infection. In some cases, the surrogate should not havea long-term persistence in the environment. It should be predictable,easily detected, and decontaminated without jeopardizing industrialequipment or natural environments. Both pathogenic and non-pathogenicorganisms have been used as surrogates for a variety of purposesincluding studies on survival (under environmental conditions and duringdisinfection), transport as well as for methods development and as“indicators” of certain conditions.

A useful surrogate must display behavior (in the product/process ofinterest) similar to pathogens, spoilage bacteria and fungi, or othertarget organisms. This includes attachment and growth under conditionsof inadequate sanitization, and complete neutralization undersanitization conditions that remove the target. This implies that thesurrogate is changed by the process. For example, a cleaning process mayeither render target microbes unable to attach, or kill them in place.Likewise, an effective surrogate may be converted into an unattachedstate by effective sanitization, or simply converted into anundetectable state.

A surrogate preferably also would have the following characteristics:

-   -   Safe (non-pathogenic, not hazardous to equipment or operating        personnel);    -   Can be detected rapidly to allow for additional sanitation steps        to be taken, if necessary, without significant incremental plant        downtime;    -   Inexpensive;    -   Easy to store, prepare, apply, sample, and detect/enumerate;    -   Easy to differentiate from residual product or background        microflora; and    -   Will not establish as a “spoilage” organism in the processing        facility.        Encapsulated DNA Sequences as Surrogates

Advances in bio-engineering have produced materials that enable thedevelopment of an efficient, effective and low cost food pathogensurrogate system. These surrogates can be formed through the use oftrigger DNA, as a DNA bar code or as a simple tag encapsulated in thecarrier. FIG. 1 is a schematic illustration of a pathogen surrogate 105formed by combining a DNA bar code 103 and a carrier material 101. FIG.2 outlines a method for determining the effectiveness of the surrogate,where the carrier is received at 201, combined (encapsulated, absorbed,and/or adsorbed) with the DNA tag at 203, and the benchmarking of thesurrogate is performed at 205. In one possible embodiment, the triggerDNA consists of a substance comprising around 90-150 DNA base pairs ofnatural or synthetic DNA. Maltodextrin, salt, starch or many other foodgrade materials may be used as carriers. Additionally, non-water-solublefood grade polymers, proteins, membrane lipids, etc. can be used ascarriers. The particle is a safe, size-selectable surrogate containingDNA that is not biologically active, but nonetheless containsinformation chemically encoded by the specific base sequence.

Measurement of DNA molecules is commonly performed through processesthat specifically amplify the copy number of only those moleculesbearing a specific target base sequence. Such processes include thermalcycling (polymerase chain reaction, aka PCR) and isothermal technologies(including loop-mediated isothermal amplification, aka LAMP). Thesemethods amplify, detect, identify, and quantitate the target molecule.For DNA molecules>60 bases in length these methods may be used tosimultaneously measure many targets using a single reaction. And suchmultiplexing can take advantage of both differences in the target basesequence, as well as lengths of the target molecules. Preparation ofsuch sets of related/similar target sequences is straightforward forsynthetic DNA. For DNA isolated from natural sources the sequence couldbe varied through standard molecular biology techniques.

Quantitation may be performed concomitant with these amplificationtechnologies, e.g., through the use of quantitative PCR or assimilatingprobe LAMP. Otherwise the detection and quantitation may be performedusing complementary technologies, such as surface enhanced Ramanspectrometry (SERS) or surface plasmon resonance (SPR).

Encapsulated short DNA sequences offer the opportunity to develop a setof surrogate particles that can behave very similarly to a correspondingset of target pathogens, which may be found in a food processing plant.DNA sequences containing 100 or less base pairs are safe and have beenapproved by the FDA as Generally Recognized As Safe (GRAS) foodadditives. The surrogate particles can be produced in large quantitiesat relatively low cost. By varying the carrier material and formulationdetails, the surrogates' physico-chemical properties may be adjustedover a wide range of particle size, hydrophobicity, porosity,solubility, etc. Thus the behavior of the surrogates, includingadherence to the product and processing equipment, can be tuned to matchthose of specific pathogens. The surrogates' resistance to variousdisinfection and sanitation agents and methods can be further adjustedby inclusion of UV inhibitors, anti-oxidants or other chemical modifiersin the carrier material.

The surrogates may be combined with antibacterial components to whichthey are resistant to provide a combination of sanitation and sanitationverification in a combined application. In certain applications, theaddition of isotopic tags, SERS-enhanced particles, or fluorescentmaterials, such as quantum dots or nano-dots to the surrogates mayenable faster detection methods. The surrogates' stability over time maybe adjusted by inclusion of oxidizers in the carrier material. Byvarying the surrogates' carrying material, their stability under varyingtemperature, humidity, and pH conditions may be adjusted. The surrogatesmay be introduced to the process via a variety of methods, as solidpowders (sols), aerosols or liquid sprays.

Different target organisms may behave quite differently in any givenprocess, and the same target organism may behave differently in slightlydifferent processes. Thus surrogates will comprise various formulationsof the DNA tracer molecules. For each target organism-process pair apanel of surrogates should be studied. Under test conditions, thesurrogates and actual target organisms should be introduced together,and then sampled and measured together, both before and aftersanitation. Some surrogate formulations will prove more labile than thetarget, and some will prove less labile. Judicious choice of surrogateformulations, measurement methods, and quantitative decision pointsshould allow selection of a proper surrogate or set of surrogates foraccurate modeling of target behavior.

For benchmarking purposes, the maximum likelihood method is useful toassess the performance of the chosen sanitation method. After this, acovariance analysis can be used to compare the pathogens' and thesurrogates' survival rates and ensure that the surrogate and thepathogen exhibit the same behavior.

Sanitation Validation/Verification

The validation/verification process can include surrogate selection,preparation, and introduction, the sanitation process, and quantitationof the surrogate. FIG. 3 outlines the procedure: at 301 one or moresurrogates are introduced, with the sanitation process following at 303,after which a detection operation is performed at 305. As for surrogatepreparation, the surrogate used for the validation is prepared in thesame way as it was prepared during the benchmarking proceduresestablishing its correlation with the target organisms.

The surrogate can be introduced into the plant in a way that does notalter the normal condition of the environment. The surrogate may beintroduced in a variety for methods, for example as an aerosol coveringlarge areas of the plant, as a suspension in a liquid spray for a moretargeted application, or on coupons that have been inoculated with knownquantities of the surrogate and placed at specific locations in theplant (as for example areas known to be challenging for sanitization).This list is not exhaustive as other methods specific to the environmentand sanitation method may be employed. Because of the very large numberof unique DNA barcodes available, differentiated surrogates of the samepathogen may be released at different locations in a plant to study thetransport of the pathogen, for example, from the raw materials area tothe shipping area. After the surrogate is introduced in the plant thesanitation process is performed per normal operating procedure.

Following exposure of the surrogates to the sanitation process, thesurviving surrogates must be recovered and enumerated, as either (a)count reduction method where the number of surviving surrogates isestimated and the log reduction can be calculated by simply subtractingthe log of the number of survivors from the log of the initial number ofsurrogates or (b) end point technique where multiple samples arecollected and the log reduction can be calculated based on the number ofunits that test positive for surviving surrogates.

Once the log reduction for the surrogate is calculated it can beconverted to the theoretical log reduction of the target pathogen(s)using the pre-established correlations between surrogate and pathogen.It can then be determined whether the control process meets the acceptedcriteria and the desired safety objective, thus achieving validation.

One application for which these surrogates are particularly suitable issimulation of microbial biofilm. In naturally occurring biofilmsmicroorganisms form colonies that adhere to solid surfaces throughmolecular and physico-chemical mechanisms that are currently poorlyunderstood and extremely difficult to model. Organisms in biofilms areunusually resistant to all methods of sanitization. To mimic biofilmssurrogates can be formulated in thick, adherent preparations ofedible/GRAS gels and gums. These gels may be prepared from agar agar,carrageenan, gellan, gelatin, latex, glycerol ester of wood rosin, orvarious other starches, pectins, and proteins, to name a fewpossibilities.

Other Applications

These techniques can be extended to a number of other applications, asvalidation is required in many settings other than food processing. Forexample, sanitation and validation are performed in healthcare settingsincluding room surfaces, garments and medical devices; in “clean”manufacturing; and on spacecraft prior to launch. The surrogate methoddescribed here is similarly applicable to all applications.

Water Wash Systems for Fresh Produce

As a salient example food safe pathogen surrogates with a DNA tag or barcode can also be used for the validation and verification of wash watersystems such as those used in fresh produce processing. As noted above,food agriculture occurs mostly outdoors, so the food products aresusceptible to contamination from the soil in which it is grown, variousarthropods that co-inhabit the environment, and excreta from birds andother animals. Thus, most fruits, vegetables, and nuts sold as freshproduce in the U.S. are washed by the packer/shipper prior todistribution. This washing is effective at removal ofcross-contamination, which may be associated with fungi and bacteriathat can cause disease or accelerate spoilage; however, microbiologicalcontamination is comparatively tenacious and may not be adequatelyreduced by washing under conditions that preserve the product. In termsof HACCP, washing is not considered a kill step. In fact, washingproduce creates a risk of inadvertent cross-contamination, as the grosscontamination breaks down and spreads microorganisms throughout thewash.

Thus, other than rinsing off perceptible filth, the major function ofproduce wash water (and wash water additives) is not to remove attachedmicrobes from the surface of the produce, but to prevent microbialdeposition from the wash water. Although many additives have beentested, the emerging paradigm suggests that chlorine is effective tokeep the clean produce clean, while preserving quality. Too muchchlorine can rapidly damage produce and/or leave an unpleasant lingeringodor. Too little chlorine risks contamination of clean material bymicrobes released into the wash water. Thus, modern produce washingfacilities often incorporate automation to maintain an optimumconcentration of active chlorine, as it is continuously consumed by thebio-organic load of produce and filth. These systems sometimes alsomanage additional additives, such as citric acid to maintain pH below6.5.

In addition to chlorine and pH, other variables affect wash efficiency.These include temperature, contact time, and agitation, as well as thevariable survival and adherence of different contaminatingmicro-organisms. Appropriate washing conditions are related tocharacteristics of the produce. For example, leaf lettuce must be gentlyfloated in cold water to prevent damage, while potatoes are bettercleaned with vigorous mechanical friction. Practically, these conditionsare already established through industry's deep experience with washingof produce. However, validation of these methods for prevention ofmicrobial cross-contamination has not been performed. In theory,reproducible wash conditions may be validated to demonstrateeffectiveness, and automation can generate a verifiably safe andwholesome fresh product.

One of the practical limitations of wash systems validation andverification is that actual pathogens cannot be taken in to afood-processing establishment to verify a specific process. Whilelaboratory research can be used as a reference point, it is not a truesubstitute for actual, in situ validation. The availability ofnon-pathogenic bacteria, referred to as surrogates, which have similarresponses to specific food processes as the pathogenic bacteria, allowsfood processors to validate a process in-plant, without the use ofactual pathogens. As mentioned earlier, however, the industry isresisting the use of live bacteria surrogates in food processing plants,whether they are considered pathogenic or not, in part because of pastexperiences with organisms that were considered non-pathogenic butcaused reported illnesses, and in part because DNA fragments from theseorganisms may be detected during routine culture-independent microbialtesting and result in presumptive positives with highly detrimentaleconomic effects.

As indicated above, produce washing faces two primary challenges. Oneproblem is that contaminants may adhere to the produce or processingequipment in the form of recalcitrant biofilms that respond in a limitedfashion to aggressive washing. The other problem is that anymicroorganisms that are effectively washed off from a spot contaminationmay subsequently adhere to clean produce, spreading a small concentratedcontamination throughout a large number of product servings. Thesurrogates described may be used to address both of these challenges.

To address the first challenge, the surrogate can be formulated in a gelor gum that mimics the behavior of a biofilm in the wash system. Thismaterial may be applied to the surface of the processing equipment, to aselected piece of produce, or to a proxy (such as a tennis ball in anapple operation). Different surfaces may require different respectiveformulations, which may be achieved through adjusting the physical andchemical parameters of the surrogate particle formulation. For example,the waxy cuticle of leafy produce may adhere well to hydrophobicsurrogate formulations, which may be produced through incorporation oflipids, while steel may adhere better to particles providing an anionicsurface, which may be produced through incorporation of organic acids orother chelators.

Properly tuned through selection and adjustment of the specificformulation, such a surrogate will partly wash away, but partly remainstuck to the surface. Thus, after the wash process is completed theoriginally contaminated item can be retrieved, sampled, and tested tomeasure the remaining DNA tracer signal. An effective wash process mightfully remove the surrogate, which mimics removal of adherentmicro-organism biofilm. Alternatively, an effective wash might simplyrender the surrogate undetectable, mimicking killing of themicroorganisms in the biofilm.

These two possible effects are not necessarily operationally equivalent,and monitoring the wash water for the presence of micro-organisms andsurrogates can distinguish between them. If the micro-organisms andsurrogates are eluted from the surface intact, then they may redepositon clean surfaces. This is especially problematic if a pathogenre-deposits onto the surface of produce. As indicated above, this is thesecond challenge faced by produce wash methods. This second challenge(re-deposition) can be separated from the effect of the first challenge(contaminant removal) by adding the surrogate to the wash water, ratherthan a washed solid surface.

After the surrogate is introduced in the system, the wash process can beperformed following normal operating procedure. Under effective washconditions, in which microbes cannot attach to produce, a surrogatesimilarly will not stick well to the produce. To some extent, this mayoccur via destruction of the target organisms and surrogate material,via reaction with chlorine and other additives. Alternatively, thesurrogate and microbes may both simply remain in the water, but notattach to the produce. This may occur through changes in the surface ofthe produce, the microbes, and/or the surrogate. On the other hand, washconditions that are too mild can allow microbes and surrogate to bind tothe produce. Then if a surrogate is detected on produce sampled afterthe wash process, it will serve as indication that the process canresult in cross contamination and therefore failed to perform itsintended function. Detected within minutes, the DNA tag will allow foraccurate validation and verification that is faster and less expensivethan other methods.

Surrogate Production

The DNA sequences of the surrogates are produced using bioinformatics,to prevent any false positives from other sources. They may be based ontheoretical molecules, naturally-occurring DNA, or based on an existinglibrary of DNA sequences developed for biodefense and food traceabilityapplications. The DNA portion of the surrogate may be producedchemically, synthetically using biotechnology (either cell-freemolecular biology or cloning into an expression bacteria or yeast), orvia isolation/purification from natural sources. The robustness of theDNA carrier material defines the resistance of the surrogate to thesanitizing agent.

For example, surrogate that is easy to destroy via sanitization can bedeveloped, based on a DNA tagged biosimulant for aerosol transportstudies. The formulation can be a maltodextrin carrier and bewater-soluble. The solubility of the material makes the DNA readilyavailable to any oxidative sanitizer, such as active chlorine, if thematerial is in a water environment. This allows for a very basic butsignificant check for cleaning procedures (i.e., was any cleaningconducted with sanitizer?).

A PLGA-based carrier can also be used. PLGAs are FDA approvedbiodegradable copolymers of poly(lactic-co-glycolic acid) commonly usedfor drug delivery. PLGA decomposes through hydrolysis, and this processis accelerated at elevated pH and oxidative conditions. PLGA hydrolysisrates are also dependent on the ratio of the lactic and glycolic acidratios. This dependence will allow the process to fine-tune thedegradation rates to match a target pathogen. The surface of PLGA isrelatively hydrophobic and may associate with surfaces non-specifically.PLGA also bears pendant carboxylic acid groups, which may be chemicallymodified to introduce a relatively low density of functional ligands.

In other embodiments, a surrogate can be produced from a gelatin-basedcarrier. Gelatin has many unique properties, most notably very lowsolubility in cold water and high solubility in hot water. Because it isa protein, gelatin provides a particle surface bearing primary amine andcarboxyl chemical moieties, which are easily modified via controllablechemical reactions, producing a relatively high density of ligands.Other temperature-dependent surrogates may also be used. Other examplesof surrogate carrier can include: carrageenan, carnauba, silica,water-soluble carbohydrate, flour, albumin, casein, a particle bearing acore of a functionally ferromagnetic material, or carrier using fixed(killed) cells of a formerly living microorganism.

A two-step mechanism to deactivate the DNA of the surrogates can beused, wherein step 1 will induce a chemical change to the carrier,causing the release of a DNA deactivating compound in step 2.

Surrogate Benchmarking

In many applications it is useful that a surrogate actually gives aslight background when no microbes are recovered. In one embodiment, amixture of three DNA tagged surrogates, each formulated for differentstability, can be used. One formulation of the surrogates can use acarrier or formulation selected to exhibit overly robust attachment tothe produce matrix. Under wash conditions that are validated to preventmicrobial attachment, this surrogate will detach from the spikedproduce, survive the wash water, attach to unspiked produce in the wash,and exhibit strong recovery from the washed produce. This component willserve as a positive control for industrial validation.

A different surrogate can be formulated as the negative control. Thiscomponent will be developed with a labile formulation, such as acarbohydrate formulation, which will exhibit no attachment whensubjected to a mild pure water rinse, while target microbes remainstrongly attached. Inadvertent recovery of this surrogate component willindicate problems with sample handling, including inoculumcross-contamination or a skipped wash step.

A third component can be tuned for low but measurable attachment andrecovery. For example, under validated wash conditions it will return abackground of 10² to 10³ copies of surrogate (and zero microbes). Aresult of more than 10⁴ copies of the surrogate thus suggests washfailure, i.e., non-verification. Higher numbers of surrogate recoverywill correlate with higher possibility and higher most probably numberof microbes (more serious failure of the wash step), the numericalrelationship between surrogate and microbe counts may not be linear.

In the determination of different formulations, unwashed spiked producecan provide quantitation of the inoculum (microbes and surrogate), as apositive control, and unspiked samples can provide a negative control.Additionally, residual wash water can be tested for microbes andsurrogate as internal controls. The ramifications of a false negativetest for sanitation are quite serious. Thus, it is often preferable tominimize the possibility of false negatives, at the expense of anincreased chance for false positives. The system can be designed anddeveloped to yield a signal that is above background in the presence ofeffective washing. An unusually low signal in a sample (between, say,zero and 10² copies of surrogate) might therefore result from unusuallystringent washing, while a low result in the positive control willgenerally indicate a failure of the surrogate system. False positives,on the other hand, are minimized by including a negative control inaddition to our low-level surrogate.

Other detection devices can be based on high-performance biosensorarrays using a CMOS (complementary metal-oxide-semiconductor)manufacturing processes. The devices based on this technology aregenerally referred to as CMOS biochips and can identify multipletargets, including nucleic acids (DNA or RNA), peptides, or metabolites,in a massively parallel fashion (10² to 10⁸ biosensors per biochip).CMOS biochips are highly integrated and include not only the moleculardetection elements, but also all the analog sensor interface, dataconverter, and digital signal processing (DSP) components. Thisintegration means that they do not require a bulky reader, scanner,delicate optics, or sophisticated microfluidics like conventionaldiagnostic instruments today. A CMOS biochip is a bio-analysisinstrument-on-a-chip.

Conclusion

Although the various aspects of the present invention have beendescribed with respect to certain preferred embodiments, it isunderstood that the invention is entitled to protection within the fullscope of the appended claims.

The invention claimed is:
 1. A method of testing an efficacy of asanitation process, comprising: providing a first surrogate for a firstpathogen, wherein the first surrogate comprises a first taggantchemically bound to a first surrogate carrier, wherein the firstsurrogate carrier is non-toxic and comprises one or more of abio-degradable polymer, maltodextrin, salt, starch, a non-water-solublefood grade polymers, a protein, a membrane lipid, a gelatin-basedpolymer, agar agar, carrageenan, gellan, gelatin, latex, glycerol esterof wood rosin, pectins, carnauba, silica, water-soluble carbohydrate,flour, albumin, casein, a particle bearing a core of a functionallyferromagnetic material, or fixed cells of a formerly livingmicroorganism, wherein the first surrogate has been determined to havedegradation rates that match degradation rates of the first pathogen,and wherein the first taggant is formed of a first DNA bar code that hasa DNA degradation rate under the first sanitation operation independentof the carrier degradation rate; applying a first amount of the firstsurrogate to one or more processing or product surfaces; subsequentlyperforming the first sanitation operation, whereby a portion of thefirst surrogate carrier is degraded by the first sanitation operation;and subsequently performing a detection operation to determine a secondamount of the first surrogate present on the one or more surfaces afterperforming the first sanitation operation, wherein determining thesecond amount is performed by determining an amount of the first DNA barcode present on the one or more surfaces; and determining the efficacyof the sanitation process from a comparison of the second amount to thefirst amount.
 2. The method of claim 1, wherein the first surrogate isapplied to one or more surfaces of a food processing facility.
 3. Themethod of claim 2, wherein the first surrogate is applied as an aerosolor a suspension in a liquid spray.
 4. The method of claim 1, whereinapplying the first surrogate includes applying the first pathogensurrogate to a food product.
 5. The method of claim 1, wherein the firstsanitation operation includes a wash water process.
 6. The method ofclaim 1, wherein the detection operation includes use of one or more ofa polymerase chain reaction (PCR), loop mediated isothermalamplification, or surface enhanced Raman spectroscopy.
 7. The method ofclaim 1, further comprising: prior to performing the first sanitationoperation, providing a second surrogate for the first pathogen, whereinthe second surrogate comprises a second taggant chemically bound to asecond surrogate carrier, wherein the second surrogate carrier isnon-toxic and selected to have a carrier degradation rate comparable toa pathogen degradation rate of the first pathogen under a firstsanitation operation, and wherein the second taggant is formed of asecond DNA bar code that has a DNA degradation rate under the firstsanitation operation independent of the carrier degradation rate;applying a first amount of the second surrogate to one or more surfaces;wherein the first and second surrogates have differing degrees ofrobustness relative to the first sanitation operation, and wherein thedetection operation is further performed to determine a third amount ofthe second surrogates present after performing the first sanitationoperation, wherein determining the third amount is performed bydetermining an amount of the second DNA bar code present on the one ormore surfaces.
 8. The method of claim 1, wherein the first non-toxicpathogen surrogate carrier includes one or more of maltodextrin, a salt,a starch, a non-water soluble food grade polymer, a protein, or a lipid.9. The method of claim 1, wherein the first non-toxic pathogen surrogatecarrier is a poly(lactic-co-glycolic acid) polymer.
 10. The method ofclaim 1, wherein the first non-toxic pathogen surrogate carrier is agelatin based carrier.
 11. The method of claim 1, wherein the firstsurrogate includes UV inhibitors.
 12. The method of claim 1, wherein thefirst surrogate includes anti-oxidants.
 13. The method of claim 1,wherein the first non-toxic pathogen surrogate carrier includes one ormore of carrageenan, carnauba, silica, water-soluble carbohydrate,flour, albumin, casein.
 14. The method of claim 1, wherein the firstsurrogate carrier is a particle bearing a core of a functionallyferromagnetic material.
 15. The method of claim 1, wherein the firstsurrogate includes fixed cells of a formerly living microorganism. 16.The method of claim 1, wherein the first DNA bar code is encapsulated inthe first surrogate carrier.
 17. The method of claim 1, wherein thefirst DNA bar code is absorbed by the first surrogate carrier.
 18. Themethod of claim 1, wherein the first DNA bar code is adsorbed onto thefirst surrogate carrier.
 19. The method of claim 1, wherein the firstsurrogate carrier comprises a food-grade protein.
 20. The method ofclaim 19, wherein the first surrogate carrier comprises anon-water-soluble food-grade protein.